Cytokinesis in Arabidopsis: Cytokinetic vesicles, cargo proteins and fusion machinery

在植物细胞质分裂过程中，分配膜（又称细胞板）是通过分泌囊泡沿着动态细胞骨架阵列（又称片胞体）定向递送到细胞分裂平面而形成的，在分裂平面上，这些囊泡彼此融合，然后与横向扩张的细胞板边缘融合。细胞板经历成熟过程，包括逐步合成和组装新形成的细胞壁。膜融合涉及特定的qa - snare（如KNOLLE或SYP132），它们与其伴侣形成跨snare复合物。细胞动力学囊泡运输什么蛋白质，它们在细胞质分裂中可能起什么作用，实际上是未知的。在当前的资助期内（JU 179/24-1），我们已经建立了方法基础，利用免疫沉淀和质谱法鉴定驻留在细胞动力学囊泡上的货物蛋白。在我们在knolle标记的囊泡，syp132标记的囊泡或两组中检测到的250个候选蛋白中，我们选择了17个由G2/ m期特异性表达基因编码的膜相关蛋白，以便在转基因植物中进行更详细的分析。(1)主要目的将是解决是否只有一个细胞动力学囊泡群体或是否有几个的问题。例如，可以想象，携带特定SNARE蛋白复合物的囊泡只运输特定的细胞动力学货物蛋白亚群。另一种可能性是，新合成的货物蛋白在其他囊泡上运输，而不是从质膜上被内吞并重新定位到细胞分裂平面的蛋白质。(2)另一个目标将是分析货物蛋白的功能或结构贡献-包括已知的和那些有待鉴定的-细胞分裂。我们将优先考虑在细胞周期的G2/M期特异性合成的膜相关蛋白。我们将产生敲除突变体，并分析它们可能的细胞分裂缺陷，使用合适的亚细胞标记或超微结构分析。此外，我们将尝试识别相互作用的蛋白质。(3)我们还将采用新的策略来鉴定细胞动力学货物蛋白。沿着这些思路，我们将尝试增加IP-MS分析起始材料中分裂细胞的比例。此外，我们将探索一个非常不同的方法。我们将筛选在G2/M期特异性表达的基因的可用数据集，以寻找编码膜相关蛋白并在其启动子区域具有MSA（“有丝分裂特异性激活因子”）元件的基因。同样，我们将主要关注功能未知的蛋白质，其序列在进化中是保守的。

In plant cytokinesis, the partitioning membrane (aka cell plate) originates through targeted delivery of secretory vesicles along a dynamic cytoskeletal array (aka phragmoplast) to the cell division plane where these vesicles fuse with one another and later with the margin of the laterally expanding cell plate. The cell plate undergoes maturation including the stepwise synthesis and assembly of the newly forming cell wall. The membrane fusions involve specific Qa-SNAREs (such as KNOLLE or SYP132) which form trans-SNARE complexes with their partners. What cargo proteins are transported by the cytokinetic vesicles and what roles they might play in cytokinesis is virtually unknown. In the current funding period (JU 179/24-1), we have established methodological foundations to identify cargo proteins resident on cytokinetic vesicles, using immunoprecipitation followed by mass spectrometry. Of 250 candidates that we have detected on KNOLLE-tagged vesicles, SYP132-tagged vesicles or both groups, we have selected 17 membrane-associated proteins encoded by G2/M-phase specifically expressed genes for more detailed analysis in transgenic plants.(1) A primary aim will be to address the question of whether there is only a single population of cytokinetic vesicles or whether there are several ones. For example, it is conceivable that vesicles bearing specific SNARE protein complexes transport only specific subsets of cytokinetic cargo proteins. Another possibility could be that newly synthesised cargo proteins are transported on other vesicles than proteins that are endocytosed from the plasma membrane and re-targeted to the plane of cell division.(2) Another aim will be to analyse the functional or structural contributions of cargo proteins – both known ones and those to be identified – to cytokinesis. We will give preference to membrane-associated proteins that are specifically synthesised during G2/M phase of the cell cycle. We will generate knockout mutants and analyse them for possible cytokinesis defects, using suitable subcellular markers or ultrastructural analysis. In addition, we will attempt to identify interacting proteins.(3) We will also pursue new strategies with which to identify cytokinetic cargo proteins. Along these lines, we will attempt to increase the proportion of dividing cells in the starting material for IP-MS analysis. In addition, we will explore a very different approach. We will screen available datasets of genes specifically expressed during G2/M phase for genes that encode membrane-associated proteins and have so called MSA ("mitosis-specific activator") elements in their promoter regions. Again, we will predominantly focus on proteins of unknown function whose sequences have been conserved in evolution.